Nucleoside-specific ribonucleases (hereinafter “RNases”) are important tools for locating the more than 120 modified nucleosides that may be found in an RNA sequence by the process generally referred to as RNA modification mapping. RNA modifications may be associated with a variety of human diseases through both structural and functional roles. Identification and location mapping of nucleoside modifications within the overall RNA sequence is important for determining the biological roles of nucleoside modifications. Traditionally, RNA-sequencing technologies primarily relied on polymerization-dependent copying of RNA into deoxyribonucleotides through Watson-Crick base pairing. However, this copying leads to a loss of modification information of the original RNA sequence.
Mass spectrometry (hereinafter “MS”) can directly measure the mass shift associated with RNA modifications. One RNA mapping approach involves hydrolysis of a target RNA to yield nucleosides. Then, MS is used to create a census of nucleoside modifications and nucleoside-specific RNase digestion of the target RNA is used to identify modification placement. Knowledge of the compositional value of one nucleoside residue imposes a constraint on the number of possible base compositions for a given mass value. Thus, to simplify the MS analysis of RNase digestion products, much effort is expended to determine the compositional value of at least one nucleoside residue. In practice, base-specific RNase digestion of RNAs followed by separation and MS using ion-pairing, reverse phase liquid chromatography, or IP-RP-LC-MS, and collision-induced dissociation tandem mass spectrometry, or CID-MS/MS, allows one to map modified nucleosides onto the original RNA sequence.
Few nucleoside-specific or nucleoside-selective RNases are commercially available. Guanosine-specific RNase T1 and pyrimidine-selective RNase A are both commercially available and compatible with MS-based RNA modification mapping. Purine-selective RNase U2 is also commercially available, but only sparingly so. However, optimal RNA modification mapping requires the generation of sufficient overlapping digestion products from multiple RNases to reduce redundancies in digestion product sequences and modification placement.
Alternative strategies for generating overlapping digestion products exist. These include partial RNase digestion, the use of non-specific nucleases, and alkaline hydrolysis. However, these strategies also suffer from certain drawbacks, including non-specificity of digestion and ineffective reaction conditions causing too much or too little digestion. These drawbacks lead to poor analytical reproducibility and labor-intensive optimization processes. Therefore, new RNases with complementary nucleoside specificity to be used in RNA modification mapping could prove useful.
RNase MC1, a member of the RNase T2 family first isolated from bitter gourd seeds, is known to exhibit uridine-specific cleavage of RNA. Further, cucumber seed derived Cusativin is known to exhibit cytidine-specific cleavage of RNA. However, these RNases are not available commercially or on a large scale.